New HPV Diagnostic Assay Presented by Dr Ivan Brukner Dr Damian Labuda Hôpital Sainte-Justine COMMERCIALIZATION AND TRANSFER ASSISTANCE PROGRAM Ministère du Développement Économique, de l’Innovation et de l’Exportation Le 26 septembre 2008 Table des matières CONTENTS OF THE GRANT APPLICATION ........................................................................................ 4 1. GRANT APPLICATION FORM ................................................................................................ 4 2. PROJECT SUMMARY MAXIMUM THREE PAGES (in French) ................................................ 4 3. PRINCIPAL RESEARCHER: PRESENTATION AND CONTACT INFORMATION ......................... 5 4. DESCRIPTION OF THE TECHNOLOGY ................................................................................... 6 5. 6. 4.1. Background - HPV as etiological agent of cervical cancer. .......................................... 6 4.2. Technology developed and scientific basis ................................................................. 7 4.3. Current development status of the technology – functional prototype ..................... 9 4.4. Scientific value ........................................................................................................... 11 INTELLECTUAL PROPERTY .................................................................................................. 11 5.1. Invention disclosure .................................................................................................. 11 5.2. Previous disclosure and/or anterior art .................................................................... 13 5.3. Freedom to operate analysis (FTO) ........................................................................... 15 5.4. Revenue sharing ........................................................................................................ 17 TECHNOLOGY DEVELOPMENT PLAN ................................................................................. 18 6.1. Identification of current stage of technology development. .................................... 18 6.2. Remaining Steps and Time to Market ....................................................................... 20 6.3. Technical and technological challenges to be met and anticipated progress........... 20 6.4. Objectives sought ...................................................................................................... 25 6.5. Type of activities to be carried out ............................................................................ 26 6.6. Work plan and timetable ........................................................................................... 32 6.7. Deliverables and result indicators ............................................................................. 32 6.8. Decision-making milestones (go/no go) for measurable and clearly identified results 33 6.9. Proposal for granting the subsidy according to the decision-making milestones reached .................................................................................................................................. 34 7. RESEARCH TEAM ............................................................................................................... 34 8. ESTABLISHMENT’S PROJECT MANAGER (In French) ......................................................... 35 9. PARTNER ORGANIZATION ................................................................................................. 36 3 9.1. Role, experience and qualifications of the partner organization in conjunction with the project ............................................................................................................................. 36 9.2. Description of the partner organization's project selection process ........................ 37 9.3. Professional who will support the valorization process ............................................ 37 10. POTENTIAL MARKET ...................................................................................................... 38 10.1. Target market ........................................................................................................ 38 10.2. Business opportunity: Cervical cancer prevention program and HPV screening. . 39 10.3. Technological competition and benchmarking ..................................................... 40 11. COMMERCIALIZATION STRATEGY ................................................................................. 44 11.1. Commercialization of the assay in Canada – Warnex partnership........................ 45 11.2. Commercialization of the assay in Africa – Continental Diagnostic partnership .. 45 11.3. Other partnership and future development ......................................................... 46 12. PRO FORMA BUDGET (in French) .................................................................................. 46 12.1. Coûts du projet de maturation technologique ...................................................... 46 12.2. Montage financier ................................................................................................. 48 12.3. Documents démontrant la nature des engagements des partenaires financiers . 48 12.4. Démonstration que les autres sources de financement possibles ont été prises en considération .................................................................................................................... 49 13. OUTSIDE OPINIONS ....................................................................................................... 50 13.1. Evaluator 1 ................................................................. Error! Bookmark not defined. 13.2. Evaluator 2 ................................................................. Error! Bookmark not defined. Letters of support .......................................................................... Error! Bookmark not defined. Annex 1. Extended patent searches and comments ........................ Error! Bookmark not defined. Annex 2. Analysis of documents cited in the PCT research report ... Error! Bookmark not defined. Annex 3. Curriculum vitae – Research team ..................................... Error! Bookmark not defined. Annex 3.1. Ivan Brukner’s CV .................................................... Error! Bookmark not defined. Annex 3.2. Damian Labuda’s CV ................................................ Error! Bookmark not defined. Annex 3.3. Maja Krajinovic’s CV ................................................ Error! Bookmark not defined. Annex 4. ............................................................................................. Error! Bookmark not defined. 4 CONTENTS OF THE GRANT APPLICATION 1. GRANT APPLICATION FORM Please see attached form. 2. PROJECT SUMMARY MAXIMUM THREE PAGES (in French) Le projet proposé par le CHU Sainte-Justine consiste à développer d’un point de vue commercial leur nouvelle méthode de sélection de sondes spécifiques, nommée « hybridation itérative ». En particulier, on propose un essai fonctionnel pour le diagnostic du virus du papillome humain (VPH) pouvant être utilisé au sein d’un laboratoire de diagnostic clinique au Québec et en Afrique du Sud. Cette approche permet le typage efficace de génomes montrant de hauts niveaux de similitudes de séquences. À l’heure actuelle, dans le cas du dépistage et du typage du virus du papillome humain (VPH), il n’existe aucun système de sondes offrant une solution de diagnostic dont l’éventail soit complet et satisfaisant. Pour parvenir à améliorer la conception des sondes d'hybridation, les chercheurs se sont attaqués au problème de leurs spécificités et leur pouvoir discriminatoire. Leur méthode peut être étendue pour générer un système diagnostique qui repose sur l'hybridation d’acides nucléiques de courtes séquences étroitement liées. Au lieu d'ajuster les conditions d'hybridation à la sonde, un ensemble de sondes est sélectionné de manière à fonctionner dans les conditions d'hybridation. Cette méthode permettant d'obtenir une détection spécifique offre une plus grande efficacité et un plus large éventail d'applications. Un ensemble de 39 sondes spécifiques pour chacun des types de VPH ciblés a été testé. Les résultats obtenus à partir d’échantillons cliniques positifs pour le VPH, y compris les six types les plus fréquents (6, 11, 16, 18, 31 et 33), montrent que ces sondes permettent la discrimination de tous les sous-types. Ainsi, les résultats démontrent que cette stratégie permet de détecter des différences de 3-7 nucléotides entre les différents amplicons / types, à température ambiante et dans un tampon PCR adapté. La preuve de concept préclinique ayant été réussie, la prochaine étape réside dans la validation à l’aide d’échantillons cliniques. Une fois cette preuve de principe obtenue, la technologie pourra alors être adaptée dans un outil de détection et de typage plus général. Le but du projet est donc de développer et de valider la technique de « l’hybridation itérative » dans le cadre d’un test diagnostic en : 1. Effectuant une validation de l’essai prototype sur des échantillons cliniques; 2. Élaborant un format de l’essai dont le design facilitera la commercialisation. L’objectif étant d’en arriver à un essai de typage du VPH qui soit simple, efficace et peu coûteux; 5 3. S’assurant le respect des exigences réglementaires afin de permettre une commercialisation rapide de l’essai; 4. Optimisant le format commercial de l’essai pour une plus grande sensibilité et une plus grande spécificité; 5. Validant la performance de l’essai commercial final à l’aide d’échantillons cliniques. Plusieurs raisons justifient que nous entreprenions ce projet, dont les avantages très concurrentiels de la méthode qui sont: 1er) Capacité d'adaptation et haut pouvoir discriminatoire: Bien que plusieurs trousses de diagnostic pour la détection et / ou de typage soient actuellement disponibles sur le marché ou décrits dans la littérature, la grande variété de techniques disponibles pour la détection et le typage des ~ 40 sous types de virus du papillome humain (VPH) au niveau des muqueuses démontre le fait qu'aucune autre d’entre elles n’offrent une solution complète à ce problème. 2e) Faibles coûts et facilité d'emploi: Il existe deux obstacles importants et inter liés ralentissant le typage universel dans la population en générale, soit la complexité technique de l'essai et le prix par dosage. Cette technique, lorsqu’intégrée à un outil diagnostic, ne nécessite ni instruments coûteux, ni personnel hautement qualifié. En outre, cette méthode offre la possibilité d'unifier les tests de diagnostic pour les différents agents pathogènes dans les mêmes conditions de réaction qui, à terme, conduira à des tests de diagnostic qui soient plus simples, rapides et moins coûteux. 3. PRINCIPAL RESEARCHER: PRESENTATION AND CONTACT INFORMATION Damian Labuda, Ph.D., D.Sc. Professor, Pediatrics Department, Montreal University Sainte-Justine Hospital Research Center, room B-607 b 3175 Cote Sainte-Catherine Montreal, PQ Canada H3T 1C5 Telephone: (514) 345-4931 ext.3586 [sec. 3282] fax: (514) 345-4731 damian.labuda@umontreal.ca Damian Labuda studied biology and biochemistry at Adam Mickiewicz University in Poznan, Poland, where he also obtained his Ph.D. and D.Sc. He received additional training in Szeged, Hungary, in Saclay, France, and in Göttingen, Germany (post-doctoral fellow of Max-Planck Institute). His early works concerned structure-function relationship in transfer RNA, origin of the genetic code, biochemistry and physicochemistry of nucleic acids. Since 1982, he continued his research on RNA structure and interactions, in Cedergren’s lab in the Department of Biochemistry, University of 6 Montreal. In 1984 he joined the Research Center of the Sainte-Justine Hospital and the Department of Pediatrics, University of Montreal, developing DNA based diagnostics program as well as research in molecular, medical, population and evolutionary genetics. Presently, he carries studies in human population genetics on the origins and the evolutionary history of human populations, the founder effects and the genetic history of French-Canadians. His laboratory is also involved in genetic epidemiology studies aiming genetic bases of complex diseases, the underlying genetic models, the identification of cancer susceptibility variants as well as genetic variants influencing the disease outcome 4. DESCRIPTION OF THE TECHNOLOGY 4.1. Background - HPV as etiological agent of cervical cancer. HPVs are genetically diverse; those infecting genital epithelium represent types with low and high oncogenic potential. Low-risk HPVs, such as types 6, 11, 34, 40 44 and others, cause benign genital warts, whereas high risk HPVs (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) lead to cervical cancer. The most frequent 16 and 18 account for about 70% of infections seen in cervical carcinoma (Bosch et al., 2002; Franco et al., 2001); HPV 16 alone being found in nearly 50% of high grade lesions. There exists a substantial degree of DNA sequence identity among types as well as between malignant and benign HPV strains. The difference between particular HPV types in their sequence segments that are relevant to the HPV molecular testing can be as small as few nucleotides (Villa et al., 2000; zur Hausen, 2000). Cervical cancer is presently the second most common cancer affecting women worldwide, and the most frequent malignancy among women in developing countries (Ferenczy & Franco, 2002). It was estimated that 493,000 new cases of invasive cervical cancer were diagnosed worldwide in 2002, representing nearly 10 % of all cancers in women (Ferenczy & Franco, 2002). Indeed, the HPV-attributable proportion of the cervical cancer is estimated at more than 95%. The relative risk associating HPV infection to cervical neoplasia is very high and increases to several hundred in the case of the persistent infection with types 16 and 18 (Bosch et al., 2002; Clavel et al., 2001; Franco et al., 2001). Once molecular pathogenesis of cervical cancer was recognized, it became clear that an accurate type-specific testing of HPV is absolutely required for the disease prevention and management, as recurrent detection of high-risk HPV types is a strong predictor of high grade cervical intraepithelial neoplasia (CIN) and invasive cancer lesions (Brummer et al., 2006; Schlecht et al., 2001; Trottier & Franco, 2005; Aho et al, 2004, Gagnon et al, 2004). Importantly, in the era of HPV vaccination, planning cervical cancer screening will inevitably have to change, emphasizing need for HPV type-specific assays. (Franco et al., 2006). Several diagnostic kits are commercially available and numerous diagnostic systems have been described. However, of significant importance is the recent study by the World Health Organization (WHO) which carried detection of 24 samples of seven most frequent HPV types, using commercial individual typing kits, such 7 as PGMY line blot (Roche), SPF10-LiPa (Innogenetics), Deg GP5+/6+ reverse line blot and DNA chip (Biomed Lab Seoul, Korea). The measurements were performed in 29 independent laboratories and 12 different countries. The overall detection rate of HPV16 was 62% and that of HPV18 was 73.9%; approximately, half of the laboratories failed to identify HPV type 6. In 2008, WHO issued recommendation guidelines for the use of “reconstructed” clinical samples and therefore make a common cross-reference among different diagnostics tests and different platforms on the identically “reconstituted” samples. 4.2. Technology developed and scientific basis Four years ago we proposed a novel and generally applicable approach consisting of the development of nucleic acid probes by selection in vitro. This differs from commonly used approaches based on the rational design of probes. Addressing the common problem of DNA diagnostics to distinguish between closely related population variants, similar strains or subtypes, we developed a novel technology that would allow generation of probes discriminating DNA targets that differ only by few sequence positions. Specific probes are selected from a random oligonucleotides mixture by a process of iterative hybridization. Repetitive rounds of forward and subtractive hybridization lead to specific pools of probes with high discriminatory power from which individual probes can be cloned. In the summer 2004, we obtained the funding from CIHR to develop this technology and show its applicability using a model system of HPV infection. The first task was to develop proposed technology. For that, we used a specific segment of the viral L1 gene of six HPV types differing by one to seven sequence positions. Starting from the initial pool of probes we obtained pooled probes, and subsequently, target-specific cloned probes. Both pooled and cloned probes were shown to discriminate well between six HPV types. (Brukner et al, NAR 2007, Brukner et al, Nature Protocols, 2007). Once the technology was developed, we extended the experiments to 39 HPV types. For the selection we used the HPV genome region corresponding to the PCR amplified HPV segment usually used (Jacobs et al., 1997; de Roda et al, 1995; Evans et al, 2005,Schmitt et al, 2006; van den Brule et al, 2002; Schmitt et all, 2008; Nazarenko et all, 2008) in the clinical diagnostics (GP5+/6+ region of the viral L1 gene). We have obtained a set of 39 probes specific for each of the targeted HPV type and efficiently discriminating against the remaining 38 types in a single experiment under the ambient temperature of hybridization (Brukner et al., J. Clinical Virology, 2007). In summer 2007 we obtained proof of principle (POP) grant from CIHR to simplify our assay and render it generally applicable in the research and diagnostic setting. We tested the probes in the reverse format (as opposed to previously used direct format) in which the array of the HPV specific probes is immobilized on solid support. This format is more suitable for diagnostic use and allows simultaneous detection of distinct HPV variants in the clinical sample. Miniaturization of the assay was achieved by single compartment 8 hybridization (as opposed to previously used 96-well format). For this, the attachment of probes was performed via streptavidin-biotin connection using SAM membranes (Promega, WI). For all testing we used full-length GP5+/6+ target oligonucleotides (and double strand amplicons derived from these oligonucleotides, mimicking thus PCR from clinical samples) labeled with [32P] for initial optimization (Fig 1). 1 1 HPV 6 HPV 16 HPV 11 HPV 18 4 5 11 HPV 31 HPV39 8 12 HPV 33 HPV 51 9 18 HPV 35 HPV 68 Figure 1. Example of HPV typing probes in reverse format. Hybridization signal between 39 selected HPV type-specific probes (each 100 pmols), y spotted on SAM membrane (7 cm x 3 cm) and particular intended targets (HPV 6, HPV 11, HPV 16 and HPV 18 on upper panel and HPV 31, HPV 33, HPV 35, HPV 39 HPV 51 and HPV 68, on lower panel) each presented in the concentration of 3 pmols per 5 mL of hybridization solution. (P32 autoradiography). Hybridizaton is performed at ambient temperatue (26+/4oC) using buffers compatable with colorimetric detection. No false positives (cross-hybriization) with remaining 38 HPV types were seen. As a result of these successful developments, we subsequently focused our assay on four most relevant HPV types (6, 11, 16 and 18). These types are the most relevant from the point of view of recently introduced national vaccination programs (duration and liability of vaccination strategy can be monitored). At the same time, we can guarantee that probes that we obtained during selection will not produce significant cross-talk with other relevant HPV types as shown in Fig. 2. 9 25 20 15 6 10 11 16 18 5 6 6 11 13 16 18 26 30 31 33 34 35 39 40 42 43 44 45 51 52 53 54 55 56 58 59 61 62 64 66 67 68 69 70 72 73 76 MM4 MM7 MM8 0 Figure 2 – Hybridization signal (y-axis) between selected 6-FAM labeled type-specific probes (HPV 6, 11, 16 and 18, using 20 pmol of each) and 39 HPV GP56 targets, where HPV types-specific numbering nomenclature is ranked in the growing order on x-axis. Each target (20 pmol) is bound to one well (using 96-well steptavidincoated Pierce plate) and hybridization is performed in 100L volume (see Brukner et al, 2007, J. Clinical Virology, for more details). 4.3. Current development status of the technology – functional prototype Processes/algorithm for the generation of spectrum of specific nucleic acid (NA) probes able to discriminate against plurality of similar targets is not yet known. When sequences that are to be distinguished are similar, the difference in their binding energy is small, restricting the window of adjustable experimental conditions, which would allow discrimination between all potentially reacting species. Finding such conditions is usually problematic in multiplex applications, when many probes and/or many targets are considered simultaneously. Moreover, if probes with such a requirement have to perform under robust conditions, the design process is especially prone to failure. Here the term “specificity” is used as an ability to discriminate NA target in the context of similar NA targets (differentiating up to 87% sequence identity), while assay is considered “robust” if stability of the assay performance within a wide range of performing conditions is preserved. Following urgent need for developing more robust and more accurate HPV typing assay, we applied this approach to obtain new generation of probes. These probes are selected to discriminate among 39 clinically relevant HPV types, based upon the previously characterized GP5+/6+ L1 segment of the HPV. In a series of hybridization steps, starting from a mixture of random oligonucleotides, we iteratively enriched mixture in 10 oligonucleotides that selectively recognize each specific HPV type out of the 39 HPV targets. (Brukner et al, (2007a), Brukner et al, (2007b). A detailed analysis of data showed clearly that, given the number of variables, the rational design of probes would not be as efficient and straightforward as selection performed in vitro. Analysis of sequences of obtained probes showed that specificity of binding between probes and targets is achieved through a fine balance between non consecutive stretches of basepairs, segments of mismatches and often accommodation of secondary structures. These combinations allow maximizing the difference in binding energy between probe-specific target and probe-unspecific target complexes. In the next phase, we optimized a reverse format of probes (Fig 3). The performance of the assay was examined using clinical samples containing HPV 16 and HPV 6. Figure 3. HPV typing of pre-characterized clinical samples containing HPV6 and HPV16 to the array of 39 immobilized type specific clonded probes (CP). (A) the arrangements of CP probes; (B) hybridization with HPV6; (C) hybridization with HPV16. Arrows indicate the orientation of the probes array. (see Brukner et al, J Clinical Virol. 2007, for more details) We confirmed specificity of our probes and stability of our novel assay at the wide range of temperatures, starting from 20°C to 28°C and in the different spectrum of nondenaturing buffers (classical hybridization buffers, as well as simple, PCR-like and colorimetric-compatible buffers). 11 Such assay performance is prerequisite for the future point-of-care medical device, contrary to the present genotyping assays, whose setting performance is not only challenging for similar NA targets, but also based on sophisticated technological platforms. 4.4. Scientific value The main advantage of our method resides in its enhanced power of identification and discrimination between multiple short nucleic acid sequences that differ by a few mutations, as it is the case with different HPV types, in a multiplex hybridization assay. In other words, instead of adjusting hybridization conditions to the whole set of probe: target pairs that we want to include in the diagnostic device, by using iterative hybridization we adjust the probes to the conditions we have chosen. Example of direct expected outcome using our HPV typing assay in clinical follow-up is to estimate HPV vaccine performance and its protective time in industrial countries where HPV vaccine program is available. It will also provide a cost-effective but accurate and easy to use diagnostic tool, an essential requirement for deployment of HPV screening program in developing countries. Finally, the methodology of probe selection is applicable to many other medical conditions where investigation or diagnosis or detection of resistant strains is based upon a differentiation of highly similar nucleic acid sequences (HIV, hepatitis virus, tuberulosis.). 5. INTELLECTUAL PROPERTY 5.1. Invention disclosure In January 2006, Dr. Brukner, Dr. Labuda and Dr. Krajinovic filed an invention disclosure at the research administration of Hôpital Sainte-Justine. The inventorship’s contribution is presented in Table 1. Inventors Îvan Brukner Damian Labuda Maja Krajinovic Status Research associate Professor Associate professor Institution % inventorship CHU Ste-Justine 45 % CHU Ste-Justine 35 % CHU Ste-Justine 20 % Table 1. List of the inventors and their contribution to the invention 12 The invention describes a new method for generating sequences of hybridization probes, suitable for multiplex target detection, even if the plurality of targets are very similar at the sequence level. The method is based on the hybridization of nucleic acids. A prototype for genotyping HPV virus was built and performance of 6 selected probes was tested and compared with complementary probes. A set of 39 oligonucleotide probes for HPV typing was generated and more recently a prototype assay was optimised for 4 probes (see section 6.1.). A provisional patent application entitled “NUCLEIC ACID PROBES, METHODS FOR THEIR PREPARATION AND USES THEREOF” was filed in the US in August 2006. We filed a complete PCT application in August 2007 and we expect to enter the National Phase in different markets in February 2009 (see section 5.3.1.). M. Serge Shahinian from the firm Goudreau Gage Dubuc in Montreal is the patent agent handling the file. The data base “Derwent World Patents Index”, where specialist editors provide a comprehensive summary of the patent contents with its advantages gives the following description of the invention: NOVELTY - Identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid comprises: (a) hybridizing the first nucleic acid with oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences; (b) amplifying the bound oligonucleotides to obtain amplified oligonucleotide duplexes; and (c) repeating hybridization in the presence of a second nucleic acid, where an oligonucleotide comprising the random nucleotide sequence can be used for discriminating the first nucleic acid from the second nucleic acid. DESCRIPTION - Identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid comprises: 1.hybridizing the first nucleic acid with a pool of oligonucleotides in a hybridization mixture, the oligonucleotides comprising a random nucleotide sequence flanked by primer recognition sequences; 2.removing oligonucleotides which are not bound to the first nucleic acid from the hybridization mixture; 3.dissociating bound oligonucleotides from the first nucleic acid; 4.amplifying the bound oligonucleotides using primers capable of binding to the primer recognition sequences to obtain amplified oligonucleotide duplexes comprising a first strand corresponding to the bound oligonuc leotides and a second strand corresponding to the complement of the bou nd oligonucleotides; 5.treating the duplexes to remove or degrade the second strand to obtain single-stranded amplified oligonucleotides; 6.repeating (a) to (e), where the pool of oligonucleotides of (a) is the amplified oligonucleotides obtained in (e) thus to obtain further ampli fied oligonucleotides; and 7.repeating (a) to (e), where the hybridization in (a) is performed in th e further presence of the second nucleic acid; where an oligonucleotide comprising the random nucleotide sequence of the further amplified oli gonucleotides can be used for discriminating the first nucleic acid fro m the second nucleic acid. 13 INDEPENDENT CLAIMS are: 1.an oligonucleotide (a) identified by the method above, (b) capable of discriminating a first nucleic acid from a second nucleic acid, where the oligonucleotide is not exactly complementary to the first nucleic acid, and (c) comprising a nucleotide sequence selected from SEQ ID NO. 1- 43, 100-104, and 116; 2.a method for detecting the presence or absence of a first nucleic acid in a sample; 3.a kit for detecting the presence of a first nucleic acid in a sample, the kit comprising the oligonucleotide; 4.a collection of two or more oligonucleotides, where the oligonucleotides comprise a nucleotide sequence selected from SEQ ID NO. 1-43, 100-104 , and 116; 5.an array comprising the oligonucleotide or the collection of two or more oligonucleotides; and 6.a kit for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, the kit comprising the pool of oligonucleotides. USE - The methods are useful for identifying an oligonucleotide for discriminating a first nucleic acid from a second nucleic acid, and for detecting the presence or absence of a first nucleic acid in a sample. The kit is for detecting the presence of the pathogen in the sample, for detection of the pathogen in the subject, and for diagnosing a disease or condition associated with the pathogen in the subject (all claimed). The methods can be used for discriminating between closely related or similar nucleic acids, and for identifying or preparing an oligonucleotide for discriminating a desired or intended target nucleic acid from other undesired or non-intended non-target nucleic ac ids. The oligonucleotides, methods, and kits may be used in analytical, diagnostic (e.g. infection of an animal, plant, or organism by a pathogen), detection, manufacturing/quality control, research, environmental monitoring (e.g. pollution/contamination of air/water/reagents) intended for use in biological systems (e.g. culture or animal systems/other materials), microbiology (detection studies of organisms difficult to c ultivate), and forensic applications. ADVANTAGE - The method has the capacity of identifying or preparing an oligonucleotide for discriminating nucleic acids, which share sequence similarities, e.g. similar nucleic acid sequences from different organisms (e.g. orthologous genes), variants (e.g. polymorphisms, different alleles) of a given nucleic acid sequence, nucleic acid sequences derived from genes belonging to the same family or nucleic acids derived from subtypes of a given organism (e. g. virus, bacteria, parasites). 5.2. Previous disclosure and/or anterior art In conjunction with Univalor, the organization that provides commercialization services to the research centre of Hôpital Sainte-Justine (see section 9), we performed a review of the prior art. The search was carried out using Delphion patent databases, and literature (PubMed) together with a web-based search. 5.2.1. Method of selection probes The concept of selecting nucleic acid sequences that specifically bind particular targets has been developed using an approach called SELEX (systematic evolution of ligands by 14 exponential amplification. However that patented method (see Gold, Table 2a) and other related methods described in the literature (c.f. Kyung 2003) do not teach the use of iterative selections for generation of nucleic acid ligands against nucleic acid targets for the purpose of genotyping or identifying/detecting nucleic acids. Therefore, our iterative hybridization method to select probes that can discriminate between closely related or similar nucleic acids is entirely novel. Patent (filing date) US5475096 June 10, 1991 376 family members Title Nucleic acid ligands Inventor (Assignee) Gold, et al. (University Research Corporation) Abstract A new class of nucleic acid compounds, referred to as nucleic acid ligands, have been shown to exist that have a specific binding affinity for three dimensional molecular targets. In a preferred embodiment the nucleic acid ligands are identified by the method of the invention referred to as the Systematic Evolution of Ligands by EXponential enrichment (SELEX), wherein a candidate mixture of nucleic acids are iteratively enriched in high affinity nucleic acids and amplified for further partitioning. Table 2a. Patent related to the method 5.2.2. HPV probes Our search revealed, that prior art taught the use of nucleic acid probes to detect HPV types. Several patents (see examples in table 2b and in Annex 1) and articles (Hwang et al., 2003; Klaassen et al., 2004; Kleter et al., 1999; Schmitt et al., 2006; van den Brule et al., 2002) describe typing systems based on PCR/classical hybridization but in all cases they rely on complementary sequences to discover HPV types (hybridization rules based on Watson-Crick base-pairing). Our probes do not rely on complementary sequences and also none of our probes sequences are disclosed or claimed in these documents. Patent (filing date) US6583278 November 14, 1996 11 family members US6265154 October 25, Title Nucleic acid probes complementary to human papillomavirus nucleic acid and related methods and kits Nucleic acid primers and probes for detecting Inventor (Assignee) Gordon, et al. (Gen-Probe Corporation) Kroeger, et al. (Abbott Abstract The present invention describes oligonucleotides targeted to HPV Type 16 and/or Type 18 nucleic acid sequences which are particularly useful in aiding the detection of HPV Type 16 and or 18 by, for instance, acting as hybridization assay probes, helper probes, and/or amplification primers. Probe sequences that are useful for detecting oncogenic HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 are 15 1996 8 family members oncogenic human papillomaviruses Laboratories) US5364758 July 16, 1992 20 family members Primers and process for detecting human papillomavirus genotypes by PCR Meijer, et al. (Stichting researchfonds pathologie) US5712092 July 7, 1994 39 family members Papillomavirus probe and process for in vitro diagnosis of papillomavirus infections Orth, et al. (Institut Pasteur) herein provided. These sequences can be used in hybridization assays or amplification based assays designed to detect the presence of these oncogenic HPV types in a test sample. Additionally, the sequences can be provided as part of a kit. The invention relates to primers and a method of detecting human papilloma virus (HPV) genotypes by means of the Polymerase Chain Reaction (PCR). The invention provides such primers and such PCR conditions that in principle any genital HPV genotype is detected. The invention enables a sensitive and reliable preselection of samples to be examined, such as cervical smears. The invention relates to human papillomaviruses HPV, particularly to HPV-DNAs isolated from papillomaviruses HPV-2d, HPV-10b, HPV-14a, HPV-14b, HPV-15, HPV-17a, HPV-17b, HPV-19, HPV-20, HPV-21, HPV-22, HPV-23, HPV-24, HPV-28, HPV-29, HPV-31, HPV-32, HPV-IP2 and HPV-IP4. The invention also relates to DNA capable of hybridizing with the HPV-DNAs or fragments thereof, to kits containing distinct groups of probes containing one or more of these HPV-DNAs or fragments thereof, and to procedures for detecting and identifying HPV in tissue. Table 2b. Patent related to our HPV assay 5.2.3. Research report on the PCT patent application In November 2007, we received the international research report and the written opinion of the international searching authority. Seven documents, including four documents published by our inventors were found to oppose our invention in terms of both novelty and inventive step (nothing was opposed to the industrial applicability of the invention). We carefully analyzed these documents and our conclusion, which was validated by our patent agent, is that none of these documents teach, disclose or can be considered as anterior art of what we claim in our invention (see Annex 2 for a summary of our analysis). In conclusion, based on our analysis of the prior art, we do not anticipate any problems for the patentability of our invention and we are confident that we would be in an excellent position to argue potential objections from patent Examiners at the regional and national phases of protection, should there be any. This confidence was validated by the patent agent handling the file. It might be important to mention that Univalor’s team has experience in patent prosecution and with interpretation of PCT written search and opinion reports. 5.3. Freedom to operate analysis (FTO) We do not see any FTO problems related to the commercial use of our new method for generating sequences of hybridization probes concerning the SELEX patent (see section 5.2.1) The SELEX process is described in two issued US Patents, No. 5,270,163 (filed Aug 1992), which claims the SELEX method itself, and No. 5,475,096 (see table 2a) which claims the use of the method to identify nucleic acid ligand for target molecules 16 “other than a polynucleotide that binds to said nucleic acid ligand,” and “wherein said nucleic acid ligand is not a nucleic acid having the known physiological function of being bound by the target molecule.” Our preliminary FTO search and analysis gives us no indication that the commercial production, marketing and use of our new assay for HPV detection would infringe the intellectual property rights of other patents. Our analysis included the US patents listed in table 2c and in Annex 1. It appears that these inventions relate mainly to the HPV DNA isolated from the appropriate strain, the probes containing these DNA sequences, and the kits containing these probes. Because the intrinsic nature of our probes is not fully complementary to any of the HPV DNA, our probes are not covered by the claims of these patents. Our position was validated by our patent agent. Note that most of the HPV types were covered by patents that are now expired or will expire in the near future. Consequently, we do not anticipate a need to license any patent on either SELEX or any specific sequence of HPV variants in order to commercialize our technology. Patent number (priority date) No. 6,391,539 (November 1984), No. 5,958,674 (November 1984), No. 5,876,723 (March 1986), No. 5,824,466 (May 1988), No. 5,656,423 (December 1990) and No. 5,981,173 (February 1996) No. 4,849,334 (June 1987), No. 4,849,332 (May 1987), No. 4,849,331 (June 1987) and No. 4,908,306 (June 1987) No. 5,057,411 (April 1985) and No. 5,643,715 (October 1988) Comments Patents that are part of the broad HPV patent portfolio developed at the Institut Pasteur that were licensed to Roche in June 2002; Patents were assigned to Life Technologies (now Invitrogen) Patents were assigned to Georgetown University. Table 2c US patents related to HPV types 5.3.1. Acquired or planned protection A provisional patent application entitled “NUCLEIC ACID PROBES, METHODS FOR THEIR PREPARATION AND USES THEREOF” was filed in the US in August 2006. That patent application claims a method for identifying and preparing probes for selective detection of nucleic acids. This method is particularly useful in discriminating between closely related nucleic acid sequences. Such a method may be used in a variety of analytical and diagnostic research and related applications. Probes selected for 39 different HPV types are covered in the patent application. A complete PCT application was filed in August 2007. Priority Application Number (Number Kind Date): US 2006822153 P 20060811 PCT Application Number WO 2008017162 Patent Assignee: SAINTE-JUSTINE UHC Inventors: BRUKNER I; KRAJINOVIC M; LABUDA D PCT filing date: 20080214 17 We plan on entering the Regional and National phases in February or March 2009 as described in the following table (Table 3). 1) 2) 3) 4) Canada (February 2009) USA (February 2009) South Africa (February 2009) ARIPO (March 2009). ARIPO is considered a regional phase covering the following countries: Botswana, Gambia, Ghana, Kenya, Lesotho, Malawi, Mozambique, Namibia, Sierra Leone, Sudan, Swaziland, Tanzania, Uganda, Zambia, and Zimbabwe. We will budget another patent filing in a territory that will be chosen between the following: Europe, Angola (a relevant territory for our financial partner in this application) and Brazil (the translation of the application into Portuguese for the Angola application can be subsequently used for the Brazil application and will reduce the cost of filing in this country by 50%). The IP protection in Canada, South Africa and ARIPO is justified by the partnership with the company Continental Diagnostic, based in South Africa (see section 14), and the interest of Warnex, based in Laval, Québec to provide HPV diagnostic services through Canada. It is also strategically important to get protection in US which is the largest market for HPV testing presently. Europe and Brazil are also two relevant territories for IP protection since vaccines clinical trials were done in these territories. However the final choice between Europe, Brazil and/or Angola will be decided in January 2009. 5.4.Revenue sharing The documents attesting to the assignments of titles, rights and revenue sharing between the researchers, the CHU Sainte-Justine Research Centre (“CHU Ste-Justine”), its commercialisation entity (i.e. Valorisation-HSJ, limited partnership) and/or the Université de Montréal (“UdeM”), shall be concluded prior to the start of the project, the whole according to the provisions of the intellectual property policies and related agreements in force between these institutions. As a precision, CHU Ste-Justine is an affiliated institution of UdeM and as such, CHU Ste-Justine and UdeM jointly own, in equal parts (50%/50%), the undivided ownership rights in any invention originally disclosed at CHU Ste-Justine by a researcher who holds an academic qualification or a faculty title of UdeM. CHU Ste-Justine and UdeM also jointly own, in equal shares (50%/50%), the institutional share of the benefits or revenues which will be allotted to CHU Ste-Justine, as mutually agreed between these institutions. The share of the proceeds of commercialization of the invention (the “Proceeds”) shall therefore be divided and paid as follows: 18 (A) fifty percent (50%) of the Proceeds shall be allotted to the researchers, which portion shall be divided between each of Îvan Brukner (22,5%), Damian Labuda (17,5%) and Maja Krajinovic (10%); and (B) fifty percent (50%) of the Proceeds shall be allotted to Valorisation-HSJ, limited partnership (the valorisation entity of CHU Ste-Justine), which portion shall be divided in equal shares (50%/50%) between Valorisation-HSJ, limited partnership and UdeM. Party Share of Proceeds Îvan Brukner 22,5% Damian Labuda 17,5% Maja Krajinovic 10% Valorisation-HSJ, limited partnership (CHU Ste-Justine) 25% UdeM 25% TOTAL: 100% Table 3. Final sharing of the Proceeds between the parties 6. TECHNOLOGY DEVELOPMENT PLAN The proposed development plan will allow optimizing and validating the efficacy of a new HPV assay and will provide us with convincing arguments that it can fulfill all principal requirements of clinical and market needs: multiplex detection, detection of different HPV types in the same patient (super infection), low production cost and ease of use. 6.1. Identification of current stage of technology development. The actual prototype kit is composed of 4 probes designed to detect 4 HPV vaccinerelevant types (6, 11, 16 and 18), including two positive controls: one reflecting presence of any HPV in the sample and the other controlling for sample DNA integrity (see Figure 4). HPV targets are 150 nucleotides long GP5+6+ DNA segments, derived through amplification or chemical synthesis. 19 6 11 100 100 16 18 100 100 UP CD 0.5 10 Figure 4: Schematic presentation of 4 cm x 2cm Steptavidin-coated Membrane (SAM, Promega) with biotinilated oligonucletide probes Optimal number of picomoles (right panel) spotted on the membrane (left panel) for each HPV probe and controls (UP, universal HPV probe, CD, control DNA designed for the control of the quality of DNA extraction HPV 16 and HPV 18 are the most frequent virus types found in cervical cancer. In fact, HPV types 16 and 18 account for 70% of HPV infections. HPV 6 and 11 are found frequently in genital and upper respiratory tract condylomas. Accurate and affordable HPV genotyping for these strains will also be in high demand for the next few decades due to HPV vaccination studies. Recently, two experimental vaccines to prevent infection with HPV 6, 11, 16 and 18 became available. HPV screening is needed here: i) to identify individuals that are eligible for vaccination and ii) to monitor the efficiency of vaccines. The Advisory Committee of Immunization Practices (ACIP, US) recommended the continuation of HPV screening protocols until (i) other type-dependent vaccines are developed and (ii) the protective time period of the vaccine is fully characterized. The advantage of our assay is in the selection of probes in the context of 39 viruses. Therefore, the probes are highly specific for these types and do not cross-hybridize with other HPV amplicons (see Figure 2). We also developed an HPV universal probe, which is designed to detect any HPV infection (Fig. 5, probe UP). This “promiscuous probe” allows for the follow-up of HPV positive cases (but HPV 16 and 18 negative ones), which is of particular interest to researchers and to public health. Despite typing only 4 viruses, all cases of HPV-positive cases could be clinically registered. Further type-specific analysis can be then performed by complementary methods. For estimation of DNA quality and quantity of a clinical sample, we also introduced a new probe hybridizing with monomer repeat of 29 adenines (A29), present in a single copy locus of human genome of well-described BAT26 amplicon (Fig. 5 probe CD). The performance of each type-specific probe and its corresponding intended targets is presented in Figure 6A. 20 6.2. Remaining Steps and Time to Market Class II, III and IV medical devices need to have Canadian licenses in order to be sold in Canada. After speaking with Sarah Chandler, Acting Head of the Regulatory and Scientific Section at the Device Licensing Services Division of the Medical Devices Bureau at Health Canada, we believe our HPV test is most likely a Class III product. We plan to validate the classification status of our device before the end of 2008(please introduce a new date 2009). This is the first step toward getting a medical device license. The regulatory process in South Africa starts with a letter to the authority mentioning the intention to file clinical information for registration of a new test. The authority can call for local clinical information and trials if deemed necessary. When the information has been submitted, questions could follow on the proof of concept and clinical validation methodology results. The whole process takes up to 3 years right now, but when the new authority steps in it might be shortened to 18 months and if fast tracked, 9 months. Our strategy is to partner with a company already established in the molecular diagnostic market who will be responsible for the final stages of our product development, including all aspects of clinical validation and regulatory affairs. We will have the opportunity to work with the Quebec company Warnex (see letter of support) to define the regulatory path for our HPV assay. 6.3. Technical and technological challenges to be met and anticipated progress 21 1) Preservation of assay specificity and sensitivity under different assay conditions Our results indicate that we will be able to develop a new format of HPV typing kit that will preserve clinical sensitivity (genome equivalent, GE, 1000 or more), but will have unique specificity features preserved in a wider-than-usual range of assay conditions. What remains to prove is that our type-specific probes will produce better assay stability than any other probes on the market. It is well known from literature data that small variations in HPV genotyping assay conditions can lead to the wrong interpretation of hybridization intensity patterns when using known commercial kits. The most critical issues are in the domain of specificity of hybridization among multiplicity of similar targets, where even a 1oC deviation from the intended temperature, or small variations in buffer conditions can be detrimental. In fact, recent data (Steinau et al., 2008) showed that even intra-laboratory repetitions with the same clinical samples (but different DNAextractions) did not produce the same results using Roche Linear Array assay (83% concordance). We believe that our HPV typing probes have inherent (sequencedependent) features which preserve stability under a wider range of assay conditions. Considering that this issue presents a major barrier to the current accuracy of hybridization-based assays, overcoming it by confirming our data in a clinical setting would be important accomplishment. As an example of anticipated progress, we recently perform hybridizations between HPV 6, 11, 16 and 18 single stranded targets (GP5+ strand) at 4 different temperatures (20, 25, 30 and 35oC) and recorded hybridization pattern, as presented on Figure 6B. While, as expected the efficiency of the assay (as measured by signal intensity) is affected by the temperature it preserves its specificity in a wide range of temperature conditions. 22 2) Optimization of a prototype assay format and developing it into its final product We already have a functional prototype assay format which will be challenged with reconstructed samples having 1,000 and 10,000 genome equivalents (GE) of different types of HPV in the background of 1,000 GE of human DNA (see Figure 7). 23 We do not foresee obstacles in detecting 1,000 genome equivalents of HPV in the hybridization assay. In fact, our present developments indicate that a hybridization assay is not an obstacle per se, since 1,000 GE of HPV 6, 11, 16 and 18 are amplifiable by PCR (using oligonucleotide targets as substrates) in sufficient quantity to guarantee detection of the hybridization signal. We will have to do HPV GP56 amplification in the context of human DNA, where we will have to satisfy “normal” PCR yield requirements (1-10pmols) and at the same time be able to perform CD PCR (i.e. produce amplicons which reflect DNA quality of sample). 24 3) PCR amplification from clinical samples Protocols designed to collect, store and purify DNA from cervical swabs are welldescribed. In addition, the expertise of Dr Gòrska-Flipot Izabella from the Hotel-Dieu diagnostics laboratory, guarantees that this challenge will be addressed in a professional, effective fashion given the long clinical experience and expertise she has in the domain of molecular diagnostics. 4) Technology transfer with the group from South Africa The present assay format is functional at Saint-Justine Hospital and we have to be sure that all parameters of protocol are well defined and universal, so the assay can be reproduced, without technical help from the person who developed the protocol. Therefore, we will establish a technological transfer procedure and a corresponding manual, which will guarantee that the assay can be independently repeated with the same quality of assay performance as originally described. Here is the summary of current 25 optimal conditions for each procedural step: (the details of protocol 4a to 4d might as well go to Appendix if you think it is a good idea) May be the whole description can be moved to Appendix (DL) 4a) PCR The 50 l volume PCR was performed as originally suggested (van den Brule, et al., 2002) with minor modifications including shortening elongation and denaturation time to 20 seconds. Yield of PCR was monitoring by loading 10l reaction mix over agarose gel and EtBr staining. 4b) Conversion of PCR product to single stranded (ss) DNA and labeling The 40L were digested for 15 minutes at 37C, with EXO1 (NEB), following enzyme inactivation (20min, 85C), and lambda exonuclease (15 min, 37C, following 20 min, 85C). Sample was passed over S25 column for buffer exchange reaction compatibility with downstream T4 Polynucleotide Kinase labeling step. The conversion of double stranded to single stranded form of PCR product was monitored by disappearance of EtBr stained bend before and after digestion. 4c) Membranes Streptavidine-coated Promega membranes (SAM, Biotin Capture Membrane, Medison, WI) were used in the following manner. The 1 l of 100pmols/l of type specific (CP) 5’ biotinilated oligonculeotide probes were manually sported (HPV6, HPV11, HPV 16 and HPV 18) on the surface of 3cm x 2cm membrane (see Figure 1 for spotting schematic). The HPV universal probes (UP) were spotted using 1 l of 0.3 pmols/l of oligonucleotide. Spotted drops were dried at ambient temperature for 5 minutes, membrane was washed in ddH2O for 1-2 minutes and pre-hybridized in 2 mL hybridization buffer SSPE (150 mM NaCl, 10 mM NaH2PO, 1.1 mMEDTA, pH 7.4), 0.75MNaCl, 70mMTris–HCl, pH 7.4) containing 1% SDS and 200 mg/ml heparin (hybridization oven, Model 400, Robbins Scientific ) for 1-12h at 55oC. These membranes were either stored at room temperature for couple of days, or immediately used for hybridization assay. 4d) Hybridization Labeling of 2-20 pmols of PCR and/or oligonucleotide was perfomed using 5 l of [32P] ATP (6000 Ci/ mmol) and 1 L of T4 Polynucleotide Kinase to a specific activity of 105 to 106 cpm/pmol, following produce manual recommendation (Invitorogen). Hybridization was carried out for 1-12 hours. The membranes were then washed with 1x SSPE containing 0.1% SDS for 10 min at room temperature and either exposed overnight at -80°C with intensifying screens, or exposed in Cyclone Storage Phosphore Screen (Perkin Elmer) for 10-30 minutes and read by Cyclones software (OptiQuant, version 4.00). 6.4. Objectives sought The overall goal of this project is to develop a functional HPV assay that will be implemented in a clinical diagnostic laboratory in Quebec and in South Africa. The specific objectives required to reach this goal are 1. Clinical validation of the prototype assay 26 2. Design of the commercial format of the assay (simple and cost-effective HPV typing assay) 3. Establish the regulatory pathway for the commercial use of the assay 4. Optimization of the commercial format of the assay for high sensitivity and specificity 5. Validation of the performance of the commercial assay with clinical samples. 6.5. Type of activities to be carried out The point by point summary given below refers to major issues needed for assay optimization as well as validation of sensitivity and specificity of assay with standardized and clinical samples. 1. Preparation of the test: Spotting of 4 type-specific DNA probes (6, 11, 16, 18) on SAM membranes (Promega); Inclusion of the positive control that is universal control positive for any HPV type; Introduction of the control of DNA extraction probe (DNA integrity) for clinical sample collection. To this end, independent PCR will be subsequently performed from reconstructed and clinical samples (see below). This PCR is amplifying single copy human genome segment known as BAT26 locus. Preliminary data demonstrating the performance of recently designed universal HPV probe are shown in the figures 3 to 8. The preliminary data using oligonucleotide mimicking BAT26 PCR product (without primers) are given in Figures 5. 2. Verification of the amplification sensitivity of standardized (in vitro reconstructed) samples. Range of genome equivalents (GE) of HPV (1000-10 000) will be tested. Multiplex PCR that is simultaneously amplifying any HPV genome and BAT26 locus from human DNA will be developed. 3. Evaluate the performance of different variant of GP56 primers. This would be done to see performance of different variant of GP56 primers in the context of different number of HPV genome equivalents and particularly in the context of different combinations of mixed infections (GE 1000, 10 000 and 100 000), spiked with human DNA (1000 GE)) and co-amplification of other HPVs except those here tested. Amplification products should enter into the yield range of 1-10pmols for all 39 types, where HPV16 amplicon (known to perform well) will be used as a reference point (producing 100% “standard” yield). 4. Amplification of DNA from 40 clinical samples for which custom HPV typing is already available. Samples from Department of Pathology of Centre Hospitalier de l’Université de Montréal will be used. Cervical biopsies were evaluated by service pathologists as cervical intraepithelial neoplasia of a different grade. Custom HPV detection was performed by PCR amplification with PGMY09/11 primers designed to amplify a product from the L1 open reading frame of a wide spectrum of HPV types. The HPV types were assigned by 27 restriction fragment length polymorphism based on comparison with known HPV sequences. Two restriction enzymes, RsaI and Dde I, were used which gave characteristic restriction patterns for most HPV types (Fig .9) (Gorska-Flipot et al. Ann Biochem Clin, 1996, 35, 66-70). To avoid confusion, we will call this assay the CHUM HPV assay. We are aware of the fact that each primer set used in amplification has its own bias toward particular type of viruses (see also Sabol et al., 2008). Therefore, we will try to minimize sequence-dependent differential effect of primers on production (Schmitt et al, 2008) and interpretation of results. 5. Analysis of the same reconstructed and clinical samples with commercial Roche HPV typing kit. 6. Comparative analysis with custom and commercial HPV typing. This analysis will be done in collaboration with Dr Eduarod Franco (Professor of Epidemiology and Oncology Director, Division of Cancer Epidemiology, McGill University), which for many years leads the study in HPV epdemilology (see letter). Cloning and sequencing of HPV PCR products will be performed in the case of discordant results. Activities which are currently performed to reach each objective are described in the following sections. 6.5.1. Clinical validation of the prototype assay As presented in section 6.1, the actual prototype kit is composed of 4 probes designed to detect 4 HPV vaccine- relevant types (6, 11, 16 and 18) including two positive controls, one reflecting presence of any HPV in the sample and second, controlling for sample DNA integrity. HPV targets are 150 nucleotides long GP5+6+ DNA segments, derived through amplification or chemically synthesized. We use of in vitro “reconstructed clinical samples” of HPV 6, 11, 16 and 18 following suggestion of Word Health organization for optimizing HPV tests. The variable input of GP5+/6+ HPV oligonucleotides or HPV plasmids is spiked prior to PCR with a constant amount of human DNA, originating from HPV negative cervical samples, to mimic molecular complexity of biological samples. In particular, range of genome equivalents (GE) of HPV (1000-10000) is added to human DNA. The concentration of human genomic DNA in reconstructed samples is comparable to the amount of DNA that is generally found in cervical scrape specimens (~106 human genomes/ml), (Quint et al., 2006). These “sample-reconstruction” experiments are allowing simulation of single and double HPV infection and controllable measure of analytical performance of our type-specific probes. In the next step, clinical samples (n=40) with known HPV status, as confirmed by the CHUM HPV assay (Fig. 9), will be used for estimating the performance of HPV assay. The results of HPV typing obtained with our probes and custom approach will be compared with the results of Roche LA HPV Genotyping Test. 28 HPV - PCR-RLFP 16 31 6 11 ? Rsa 16 31 6 11 Figure 9. Schematic presentation of the CHUM HPV assay (a custom PCR-RFLP used for HPV typing) 6.5.2. Design of the commercial format of the assay There are multiple formats of multiplex hybridization kits on the market and custom assays in research or clinical laboratories. Several options for commercial format exist for our technology including solid support for DNA probe attachement and for hybridization signal detection. As previously stated, our prototype assay cannot reach the market in its current format, with its large SAM membrane, radioactive detection (P32), and high cost of production. As presented in Table 4, our rough estimation of the cost of production per kit is $26 which is highly expensive for such a kit. It should be noted that our kits have actually been manufactured in the researcher’s laboratory and there is no economy of scale. The membrane cost is clearly the main issue if we want to substantially reduce production cost. Materials Oligo probe 6* Cost/ kit (CDN$) comments 0.04 umol scale needs 100pmol per spot 29 Oligo probe 11* Oligo probe 16* Oligo probe 18* 0.04 0.04 0.04 umol scale needs 100pmol per spot umol scale needs 100pmol per spot umol scale needs 100pmol per spot Two DNA control oligos to be spotted 0.08 umol scale needs 100pmol per spot Membrane SAM-Promega 25.00 $200 per membrane/8 HPV kits of 2cmX3cm 0.08 reduce background signal; increase ratio signal/background Blocker (needed to block membrane) Technician time to prepare membrane ($50/hr) - cut membrane, spot DNA, etc) Package (plastic ) total= * 67 mer single strands with biotin 0.50 0.50 26.32 100 membranes prepared per hour plastic 15mL Falcon tubes Table 4. Cost breakdown for the current production of our HPV prototype assay Our data obtained so far shows that other membrane-based hybridization (Immunodyne ABC membrane, Pall) could replace SAM membranes (Promega). Preliminary data showing performance using modified streptavidin coated Immunodyne ABC membranes are given in Fig. 10. This might be an excellent alternative for reducing the cost of the final assay given the 100 fold lower price of Immunodyne ABC versus SAM membranes. Immunodyne ABC membrane (Pall) Example of HPV 18 6 11 16 18 UP CD Figure 10. Example of possible reduction of final product prize (100x) using Immunodyne ABC-Pall instead SAM-Promega, Immunodyne membrane can replace SAM (Promega) membranes used in the experiments shown in previous slides reducing thus the most significant material cost by 100 times (20$ to 0.2$) 30 Development of an alternative system of visualization of probe–target hybridization is also essential for the simplicity and commercialization of the assay. One of the possibilities is a colorimetric assay with the use of 6-FAM labeled primers to produce PCR and a corresponding anti-6FAM-antibody with downstream alkaline phosphatecoupled colorimetric detection. Another important aspect will be to reduce the size of the assay. A 10X reduction of hybridization volume and corresponding solid support surface (beads, or array) will reduce PCR yield requirement to 50-100ng, typical for PCR volumes of 10uL. Detection of hybridization will be adequately adjusted. For example, the Luminex platform would require Cy3 fluorophore, while a slide micro-array format would allow use of DIG (digoxigenin-modified nucleic acids), or 6-FAM and the corresponding horseradish peroxidase-conjugated anti-fluorescein, or anti-DIG antibodies (Roche, Invitrogen and number of other suppliers). It is worthwhile mentioning that gold-nanoparticles are the simplest alternative for colorimetric detection of DNA (post-PCR) with robust chemistry and existing expertise in Canada (Yingfu Li, Department of Chemistry and Department of Biochemistry and Biomedical Sciences, School of Biomedical Engineering, McMaster University). In order to define the best format for the assay for commercialization, a deep analysis of the available options will be done. We plan to outsource this activity to a consultant with strong expertise in molecular diagnostics. This consultant has yet to be identified, but one possible candidate is Dr Yvan Côté, VP at Warnex, who has not only industrial experience but also who has expressed his interest in performing this analysis as a private consultant (independently from Warnex). It is worthwhile to mention that Yvan Coté has suggested that Warnex provides support to help in the design of the final format of the assay (see letter of support). Briefly, the analysis will have to take into account the scientific aspects (specificity & sensitivity), the commercial aspects (cost, license from third parties, others), as well as the regulatory requirement for the commercialization of the assay in Canada and South Africa. At least three meetings will be held with a consultant over the 8 week mandate. Participating in the meeting will be the inventors, Anne-Marie Larose from Univalor, and one representative from our potential commercial partners (Continental Diagnostic and Warnex). Meetings will include: 1) An initial meet and greet of the inventors, Anne-Marie Larose from Univalor, one representative from each of our potential commercial partners (Continental Diagnostic and Warnex). 2) A follow-up meeting midway through the mandate. 3) An oral presentation of the final report and recommendations (a written report will also be produced). 31 Experimental testing of the new format design will be performed before the beginning of the second phase of the project. 6.5.3. Establish the regulatory pathway for the commercial use of the assay The objective to get clinical validation of our HPV assay, in a commercial format, is to conclude licensing agreement with commercial partners. It is clearly not the scope of this project to get regulatory approvals in Canada or South Africa. However, we consider it is important to establish what the regulatory requirements to reach the market are. These aspects have to be taken in account for the final design of the assay and in the licensing negotiation (will help both parties to better define license milestones). This task will be driven by Anne-Marie Larose from Univalor. 6.5.4. Optimization of the commercial format of the assay for high sensitivity and specificity The outcome of phase 1 (first 20 weeks, i.e. five months) will define the choice of technological platform. Sensitivity and specificity of the assay will be re-assessed on the new assay format. This optimization step will required the following: 1) Evaluation of the right amount of DNA probes to be spotted on the new support 2) Optimization of the hybridization conditions to reduce background 3) Optimization of the signal detection 4) Development of a new reproductive procedures to perform the analysis, from the PCR product to the detection 5) Optimization of the control probes, including the HPV control probe Since we anticipate a 10X reduction of hybridization volume and corresponding solid support surface (beads or array) this will allow reduction of total PCR yield (50-100ng), typical to the volume of 10uL. At this phase of the project, technique for visualization of the hybridization signal will be adjusted to best serve the chosen platform. Comparison with results obtained with the prototype assay will help the development and optimization of the new format of the assay. Optimization will be performed with the in vitro reconstructed sample, before testing clinical samples (see section 6.5.5). Some testing will also be conducted in at least one external laboratory, ideally by one of our commercial partners, in order to validate the reproducibility of new protocols. These experiments are not covered by this proposal and will be performed at the company’s expenses. 32 6.5.5. Validation of the performance of the commercial assay with clinical samples. The performance (sensitivity and specificity) of the probes using the new platform will be compared with the typing results obtained in the first phase of the project. More precisely, the commercial format of the test will be used for HPV typing of the same 40 samples tested with our prototype assay, as described in section 6.5.1. Additional 100 clinical samples that are already charactrized by commercial tests like Roche or Hybrid Capture will be tested in this phase. The samples will be obtained through collaboration with Dr Francis Coutlée, Molecular Virology Laboraty at the Research Center of CHUM (see letter) who were running and partipating in variety of projects addressing biology, epidemilogy and diagnostics of HPV. The concordance analysis will follow. 6.6. Work plan and timetable The maturation project will require 12 months for its completion. As illustrated in table 5 the activities to meet the first objectives will start immediately while the activities for the second and third objectives will be performed concomitantly at the end of phase 1, most likely from week 10 to week 32. The activities for the last two objectives will be done successively. Dr Damian Labuda, Ms Sylvie Cossette and Dr Anne-Marie Larose, respectively the principal investigator with the inventors, the project manager and the representative of the partner organization, will meet on a regular basis, every 3 months to evaluate the progress of the project and the achievement of the milestones. The same committee will meet if necessary to evaluate any project of disclosure generated within the framework of this project in order not to reveal information that could preclude the filling of patent application. Phase 1 - 32 weeks Objectives/Activities 1 Clinical validation of the prototype assay 2 Design of the commercial format of the assay Establish the regulatory pathway for the commercial use of the 3 assay Go/No go decision 4 Optimization of the commercial format of the assay 5 Validation of the performance of the commercial assay Table 5: Gantt Chart for work plan 6.7. Deliverables and result indicators Phase 2 - 20 weeks 33 At the end of phase 1 we should have reach the first 3 objectives: 1. Clinical validation of the prototype assay 2. Design of the commercial format of the assay (simple and cost-effective HPV typing assay) 3. Establish the regulatory pathway for the commercial use of the assay More specifically this means that we will get specificity and sensitivity data with clinical samples with the prototype assay and obtain the following results: Ability to detect HPV 6, 11, 16 and 18 alone (1-10 pmols) or in a combination, where individual types are presented in final PCR product in the low picomolar ranges. No cross-hybridization with non-specific probes, under 10 pmols of one HPV types (from reconstructed samples) which represent a scenario where PCR yield is very abundant (10 pmols in total is around 1000 ng of GP56 PCR). Universal HPV probe will detect any HPV type present in PCR, while control DNA (CD) probe would be indicator of clinical sample DNA quality. The design of the final format of the commercial product will need to be defined as well as the main regulatory requirement for the commercialization of the HPV kit. We will also need to get recommendations for the final design of the project. At the end of the second and last phases of the project we should have reach the 4th and 5th objectives: 4. Optimization of the commercial format of the assay for high sensitivity and specificity 5. Validation of the performance of the commercial assay with clinical samples: Anticipated results in terms of specificity and sensitivity should be at least as good as what we get at the end of phase I with the prototype assay: specific detection of the 4 types of HPV, no cross-hybridization and positive results with the Universal HPV probes and de CD probe. Importantly, our comparative study should demonstrate that the performance of our assay is as good or superior to the Roche assay. The stability of both assays (reproducibility) using repeated typing of critical samples with PCR yield approaching 2 extreme situations (very low and very high yield) will be performed. We expect that this comparative test produce more stabile typing results with our assay format, then with Roche assay. Implementation of the new assay format in at least one clinical laboratory in Quebec, or South Africa will be performed. 6.8. Decision-making milestones (go/no go) for measurable and clearly identified results At the end of phase 1, the project will be pursued only if we get positive results on clinical samples with the current prototype (SAM membrane and P32). Herein, clinical 34 sample will have to give yield of GP56 PCR product in the minimal range of 100-1000ng (150nt long), which is in fact 1-10 pmols in total, the quantity typically detected in our present developmental experiments. This is, according to our observation and literature data, an average PCR yield of 50uL reaction volume of “classical” GP56 PCR after 40-45 cycles. We know that present solution for GP56 primers perform well for majority of HPV types, using 39 artificial targets. If our hybridization assay does not work in this range of concentrations we will consider that there is a significant failure in the process which will justify first NO GO. 6.9. Proposal for granting the subsidy according to the decision-making milestones reached Total disbursement Valorization-HSJ MDEIE Phase 1 32 weeks Phase 2 20 weeks End of phase 2 114 000 $ 70 000 $ 16 000 $ 25 000 $ 15 000 $ 89 000 $ 55 000 $ 16 000 $ Table 6. Proposed distribution of granting 7. RESEARCH TEAM In 2004, Ivan Brukner (see CV in Annex 3) joined our team to develop the iterative technology in its practical application focusing on the small sequence segments. His knowledge in nucleic acid chemistry matched the expertise of other members of the team in the physical chemistry of nucleic acids, in diagnostic application, in the development of genotyping tools, in the genetic epidemiology and pharmacogenetics, and importantly in the HPV DNA testing and in the epidemiology of HPV infections. Damian Labuda (see CV in Annex 3) has considerable experience in physical chemistry of nucleic acids, in genetic diagnostics, genetic epidemiology and in vitro selection. Ivan Brukner is the principal inventor of the proposed technology; he will be responsible for the technology implementation and daily follow-up of the experiments. Maja Krajinovic (see CV in Annex 3) has experience with the HPV DNA analysis, as well as in genetic epidemiology and pharmacogenetics; she will be involved in design of reconstructed sample experiments, selection of clinical samples (with Izabella Gorska-Flipot) and between tests concordance analysis. Izabella Gorska-Flipot (see CV in Annex 3), at the Hospital Hotel-Dieu, has a longstanding experience in the molecular diagnostics and in the HPV cervical infections in particular; she will be responsible for the analysis of clinical samples using custom approach and commercial kit. Our achievements are summarized in 4 published manuscripts Nucleic Acids Research, 2007; Journal of Clinical Virology, 2007; Nature Protocols, 2007 and Int. J Cancer., 35 2008. In addition, intellectual property related to our technology is protected. The present application is to finalize the prototype of the HPV diagnostic device and to validate its use in the clinical setting. 8. ESTABLISHMENT’S PROJECT MANAGER (In French) Gestionnaire de projet au Centre hospitalier universitaire Sainte-Justine (CHU Sainte-Justine) Le CHU Sainte-Justine est un des plus grands centres hospitaliers universitaires pédiatriques du Canada. Il a pour mission d’améliorer la santé des enfants, des adolescents et des mères du Québec. Afin d’arriver à assumer pleinement sa mission et surtout son rôle en tant que centre universitaire d’enseignement et de recherche, le CHU Sainte-Justine compte sur l’excellence de la recherche de ses chercheurs regroupés dans son Centre de recherche. Au cours des dix dernières années, le Centre de recherche du CHU Sainte-Justine a connu une croissance sans égal parmi les dix-neuf centres et instituts de recherche subventionnés par le FRSQ. Depuis sa fondation en 1973, le Centre de recherche s'est transformé en un réseau d'axes de recherche pluridisciplinaire allant de la recherche biomédicale à la recherche clinique en passant par la recherche sur les soins et le système de santé, la santé des populations et l'évaluation des technologies. Le leadership du CHU Sainte-Justine en recherche est aussi fondé sur un partenariat actif et engagé dans des réseaux de recherche. Depuis 2003, 30 nouveaux chercheurs ont été recrutés portant le nombre de chercheurs à temps complet à 90. La progression du nombre d'étudiants qui est passé de 276 à 402 au cours des cinq dernières années témoignent du pouvoir attractif du Centre, de la qualité de ses chercheurs et de sa mission de former du personnel hautement qualifié au niveau académique de même que pour l'industrie biomédicale et pharmaceutique. Les publications témoignent de notre productivité scientifique comme centre de recherche. De 2004-2005 à 2007-2008, le nombre d'articles avec comité de pairs a augmenté annuellement de façon constante passant de 364 à 385. Parallèlement à cette productivité scientifique du Centre de recherche, le CHU Sainte-Justine aura été le foyer d'une importante activité en valorisation de la recherche et des connaissances dans le domaine de la santé. Au cours des cinq dernières années, plus d'une trentaine de chercheurs ont soumis une soixantaine de déclarations d'inventions menant à trente-deux demandes de brevets, aboutissant à une dizaine d'accords commerciaux, dont neuf licences et la création d'une entreprise dérivée. Également, le Centre de recherche a assisté à une croissance continue des contrats de recherche en partenariat avec l'industrie et autres centres d’enseignement, totalisant plus de 100 nouveaux contrats en 2006 dont la valeur atteint plus de 22 millions de dollars, témoignage tangible de la valorisation du savoir-faire et connaissance de la communauté des chercheurs du CHU Sainte-Justine. Le Centre de recherche, sous la direction du Dr Guy A. Rouleau depuis près de trois ans s’est doté d’une structure administrative qui facilite le support à la recherche et la valorisation des résultats. Sous la direction de Madame Sylvie Cossette CA, adjointe au directeur et comptant plus de 15 ans d’expérience comme gestionnaire au Centre de recherche, deux professionnels spécialisées en ententes de recherche et en valorisation sont à la disposition des chercheurs. De plus, un chercheur clinicien est délégué par le Centre de recherche pour siéger sur le comité d’évaluation des technologies de sa société de valorisation Univalor. Madame Cossette sera la gestionnaire de projet de maturation technologique du CHU SainteJustine et sera responsable de s’assurer de son bon déroulement. Elle devra notamment : 36 S’assurer que l’équipe de chercheurs et le partenaire financier respectent leurs engagements; S’assurer que les objectifs poursuivis dans le projet soient en harmonie avec la stratégie de commercialisation; S’assurer avec les chercheurs que toute nouvelle propriété intellectuelle développée dans le cadre du projet soit protégée adéquatement et avec diligence; S’assurer que les ententes à intervenir entre les partenaires soient conformes aux politiques institutionnelles. 9. PARTNER ORGANIZATION 9.1. Role, experience and qualifications of the partner organization in conjunction with the project Gestion Univalor, Limited partnership ("Univalor") is a limited partnership whose mission is to commercialize discoveries made by researchers at the Université de Montréal, École Polytechnique de Montréal, CHU Sainte-Justine-Mother and Child University Hospital Center, HEC Montréal, Hôpital Maisonneuve-Rosemont, Hôpital du Sacré-Coeur de Montréal, Institut de recherches cliniques de Montréal and the Institut universitaire de gériatrie de Montréal. Univalor also provides commercialization services to the limited partnerships of the Montréal Heart Institute. Univalor strives to develop profitable and long term business relationships with companies wishing to maintain or improve their competitive position in their industry by having access to cutting edge technologies developed by internationally acclaimed researchers. Univalor's multidisciplinary team is made up of 12 professionals in business development and commercialization with expertise in life sciences, engineering, intellectual property, legal affairs, and finance. Achievements Between 2001 and 2008, about 520 discoveries from Université de Montréal and its affiliated institutions have been evaluated by Univalor and more than 900 patents applications have been filed. Univalor currently has approximately 265 patents and patents pending in its portfolio, has signed over 33 license agreements and has been involved in the creation of 23 spin-off companies over the years. Since 2001, funding provided for commercial maturation from Valorisation-Recherche Québec and the Ministère du Développement Économique, de l’Innovation et de l’Exportation (“MDEIE”) have allowed many technologies to reach maturity and demonstrate operational capabilities. More than 8.2 million dollars have been invested in 37 our most commercially promising technologies and 130 million dollars into our spin-off companies. 9.2. Description of the partner organization's project selection process Over time, Univalor has developed its own methods and tools for evaluating technologies emerging from research laboratories and for helping researchers who wish to commercialize their inventions. Internal evaluation is conducted by the business development manager and the project leader using 5-point criteria: scientific and technological quality, intellectual property, market size, business opportunity, and relevancy for Univalor. Different databases such as Dialog Pro Competitive Intelligence, Medtrack, and Delphion are used to accomplish this evaluation. Meetings with the inventor(s) and a literature review are also very important steps in order to thoroughly evaluate the technology on its scientific basis. Results of the internal evaluations are discussed weekly during our evaluation committee, which is comprised of all of Univalor’s professionals. This allows us to take full advantage of our multidisciplinary team. Relevant questions regarding intellectual property, technical issues, time-to-market, etc. often arise. The manager of business development may either pursue the evaluation after this meeting or retain the services of one or more experts. These experts, skilled in the specific technology, are asked to give their opinion about the technology on both a scientific and commercial basis under a confidential agreement. If the technology is recommended for commercialization, a technology transfer strategy is then drawn up by the evaluation committee and rapidly implemented. When a technology shows a real commercial potential, Univalor’s business development manager, along with the help of the researcher and the research administrator at the institution, looks for financial resources to perform proof of concept or validation when the development status of the technology is not mature enough to start licensing discussions with a company. Once a grant is procured and the maturation project is started, the manager of business development carefully follows the progression of the project. Results are communicated to potential partners if discussions have already been initiated and interest in the technology has been shown. Business development activities start (if they have not already started) once the maturation project begins and intensifies once the proof of principal is accomplished. 9.3.Professional who will support the valorization process Anne-Marie Larose, Ph.D, MBA, Business Development Manager, is currently responsible of this technology at Univalor. Ms. Larose has more than ten years of experience in the life sciences industry. After having obtained her doctoral degree in cellular and molecular biology, she joined a young biotech company at the pre-startup 38 stage. During the five years that followed, Ms. Larose was directly involved in the different aspects of the company's corporate and technological development, notably in the elaboration of business and commercial strategies, the protection of intellectual property, the planning and management of R&D projects, and the management of the quality control department. Afterwards, as commercial attaché for the British consulate in Montréal, Ms. Larose was responsible for commercial exchanges with the Canadian biopharmaceutical sector. More recently, Ms. Larose has contributed to the fulfillment of various industrial and institutional development mandates in the Life Sciences sector as main advisor in an advisory office. As of March 2004, Ms. Larose has been developing business relations with corporations in the Life Sciences field who wish to commercialize technologies with the greatest growth potential. 10. POTENTIAL MARKET Why typing HPV in Canada now? There are few distinct, but inter-related reasons why having “home”, provincial, or national DNA HPV typing assay, presents advantageous situation over not having it. Commercial impact is for example one of the resons. In Canada, Roche is the only assay approved by Health Canada with prize of 80-100$CA per screening (material cost), while FDA approved HC2 assay is reaching value of 42$ per single patient (QIAGEN Product Guide, 2009, listing prices, for high volume request QIAGEN offers around 50% price off). Both assays are developed in US. In Europe, the situation is more complex and diversified with more small companies trying to get part of the market. On the contrary, to our knowledge, Canada does not have any force to address this challenge. Another important factor is timing, as: 1) HPV vaccination era requires more rigorous screening activity with new and specific HPV typing tests, as suggested by Canadian and international authorities (see Rogosa et al, (2008). The urgent need for new HPV screening guidelines and new biomarkers is also well documented (Kiviat et al., 2008). The current and future necessity and role of HPV screening in the era of HPV vaccination is also well described by Myers et al., (2008). All these arguments are pointing out that HPV screening has to be continued in the new form - if not inforsed by government, because of the following reasons: 1) Vaccine is not clinically tested on already infected women, 2) The mother-child frequency of transition is not known, 3) The vaccine protective time is not yet defined and 4) 30% of cervical cancer will not be protected by the vaccine. Target market Sugestion:I would make few comments about local Montreal-Quebec-Canada market, including private laboratories, clinics, hospitals and general public awareness for diagnostics of STD. 39 The US molecular diagnostics field is the most dynamic segment of the in vitro diagnostic (IVD) market, with an expected growth rate of approximately 17% through 2012. Infectious disease testing accounts for the bulk of this market, and HPV testing is one of the fastest growing. As the second most prevalent molecular test, HPV testing has been estimated in 2006 to have reached approximately 55 million tests that year alone, representing an IVD market size of $500 million (Frost & Sullivan, 2006). Recently, the company Third Wave Technologies has provided projections which suggest that the global market for HPV testing could reach $250 million in 2008 with a market penetration of only 28% but a growth rate in excess of 25%. This includes more than 10 million HPV tests being performed in the United States each year. Finally, the HPV testing market in the EU is just emerging, as studies are underway to evaluate the use of HPV tests as a primary screen for cervical cancer in women, replacing Pap testing. 10.1. Business opportunity: Cervical cancer prevention program and HPV screening. Cervical cancer is the second largest cause of cancer deaths in women worldwide. The estimated prevalence of HPV infection in women is 10.5%. Persistent infection with HPV is a trigger for cervical cancer, and preventing this infection can avert this deadly disease. The tests used in routine screening and clinical prevention of cervical cancer are so far based mostly on a cytological examination known as the Papanicolaou (Pap) smear. In 2005, more than 60 million Pap smears were performed in the US, and it is estimated that such screening programs and interventions have reduced the incidence of cervical cancer by ~80% in the United States, but at a cost of more than $6 billion US a year (Wu et al., 2006). Although the application of the conventional Pap test led to a dramatic reduction in the incidence rate of cervical carcinoma in the last fifty years, this test has significant limitations, including a sensitivity of only 50% to 60% in a routine screening setting (Fahey et al., 1995; Nanda et al., 2000). Because of this, an improved cytological test has been developed called the thin-layer liquid based cytology, or ThinPrep Pap test. This new test is far from infallible, failing to detect from 15% to 35% of cervical intraepithelial neoplasia (CIN) or cancer (Kulasingam et al., 2002). Due to the marginal clinical benefits of liquid based cytology (ThinPrep Pap) and the major cost increase with the adoption of this technology compared to standard Pap smear testing, the Joint Technology Assessment Unit of MUHC and CHUM recommends that both institutions NOT switch to the systematic use of liquid-based cytology at this time. http://www.mcgill.ca/files/tau/Liquid_Based_Cytology_2008.pdf Recently published results of ASCUS/LSIL Triage Study for Cervical Cancer (ALTS) underline the importance of HPV DNA testing to improve specificity and reduce costs (ALTS, 2000; Sherman et al., 2001; Solomon et al., 2002a; Solomon et al., 2001; Solomon et al., 2002b; and http://www.cancer.gov/prevention/alts/results.html). Likewise, accurate and affordable HPV genotyping will be in high demand for the next few decades for ongoing HPV vaccination studies. The US Food and Drug Administration (FDA) approved the first preventive HPV vaccine on June 8, 2006 for the immunization of women between 9–26 years of age (Merck's quadrivalent vaccine 40 Gardasil, targeting HPV 6, 11, 16 and 18 ). A second vaccine, Cervarix from GlaxoSmithKline (targeting HPV 16 and 18), is already available in Australia and in Europe. So far, these vaccines have shown to offer 100% protection against persistent homologous HPV infections (Stanley et al, 2006). In the current experimental phase of vaccine trials, HPV screenings are needed to: i) identify HPV status of individuals before vaccination and ii) monitor the efficacy of vaccines. Furthermore, the Advisory Committee of Immunization Practices (ACIP) recommends continuation of HPV screening protocols until other type-dependent vaccines are developed and until the protective time period of the vaccines has been fully characterized. In spite of the above recommendations and the introduction of a number of commercial HPV genotyping kits (see table 1), a DNA test of satisfactory specificity and sensitivity to detect HPV types is still unavailable. 10.2. Technological competition and benchmarking Available HPV DNA tests, sensitivity and specificity. Key industry participants in the molecular infectious disease diagnostics market in North America are Roche, Gen-Probe, QIAGEN (QIAGEN-Digene), bioMerieux, and Becton Dickinson. QIAGEN-Digene is the sole provider of molecular HPV tests in the US. The company offers two HPV tests which distinguish two groups of HPV subtypes (benign and malignant) and offers no subtypes differentiation. The Roche HPV genotyping test (LA) and the new Genomica CLART® HPV2 test, are the only commercially available test kits for the detection of 35 or more variants of HPV. The Roche test was expected to hit the US market in 2008, but is still unavailable to date (Frost & Sullivan, 2006). A recent WHO international collaborative study (Quint et al., 2006) addressed the question of standardization of HPV molecular typing and detection. The methodology used in this study was performed by twenty-four participating laboratories included QIAGEN-Digene Hybrid Capture II, and by a variety of PCR/hybridization typing systems. The results demonstrate that the sensitivity of detection and the specificity of typing varied considerably among participating laboratories working with the eight most frequent high-risk types (16, 18, 31, 33, 35, 45, 52 and 58) and one low risk HPV 6. The sensitivity and specificity of the detection of HPV 16 and HPV 18 was only 62% and 74% respectively. For the other seven types, the adequate assessment varied from 95% (HPV 33 and 45) to 43% (HPV 31). Some false positive results were also reported. Clearly, an alternative approach to HPV typing is needed. Existing HPV DNA analyses rely either on hybridization techniques using type-specific HPV DNA probes, or are based on direct identification of HPV DNA PCR-products (see table 7). Several hybridization based tests and their prototypes have been developed that either require the viral DNA to be pre-amplified by PCR (Innogenetics, Roche Diagnostics, Genomica, Greiner Bio One) or do not require it (QIAGEN-Digene and Ventana). The main 41 difficulty of hybridization approaches that use “classical probes” in these tests, is obtaining specific results when multiple similar targets have to be assayed simultaneously (note that here we use the term “classical” to distinguish from probes obtained by our method of iterative hybridization). Probes that fully match their targets (Roche, Genomica, Greiner Bio One) compromise accurate detection of multiple targets because they cross-hybridize ((Sandri et al, 2006) and our unpublished observations). Another group of hybridization-based tests that does not require prior amplification of HPV DNA include QIAGEN-Digene’s Hybrid Capture 2 (HC2) assay and Ventana’s Inform HPV. QIAGEN-Digene’s FDA-approved assay is widely used in clinical studies due to its relative simplicity and sensitivity. However, several recent reports pointed out two main disadvantages with HC2, questioning its use as a screening tool. First, it lacks individual HPV type identification. Second, there is a significant degree of cross-reactivity between the oncogenic and non-oncogenic types that leads to false-positive results 10% to 20% of the time (Cox et al., 2000; Gravitt et al., 1998; Poljak et al., 2002; Schneede et al., 2001; Terry et al., 2001; Yamazaki et al., 2001). In brief, the Ventana Inform HPV in situ hybridization assay seems to be more specific and sensitive than HC2, but its efficacy in predicting cervical lesions (positive predictive value) is no more than 48% (Qureshi et al., 2003). Several companies offer PCR-based kits (see table 3) like GenoID which became a small participant in the European molecular diagnostics market (Frost & Sullivan, 2006). It should be noted that another option exists for the sequencing of HPV PCR products. This is performed by Visible Genetics (Toronto) (Mahony et al., 2003) and is also an inhouse test at the Hôpital Hôtel-Dieu de Montréal and at the Hôpital Sainte-Justine de Montréal. Although these sequencing tests are highly specific, they require trained personnel, expensive technology, and most significantly, are rendered useless when multiple variants of the virus are present in the same patient. Numerous other typing systems have been described (Hwang et al., 2003; Klaassen et al., 2004; Kleter et al., 1999; Schmitt et al., 2006; van den Brule et al., 2002, Schmitt et al., 2006) and they await a detailed clinical evaluation. Competitive advantages The main advantage of our method resides in its enhanced power of identification and discrimination of multiple short nucleic acid sequences that differ by a few mutations, as with the different HPV types in a multiplex hybridization assay. Because we select the most specific probes in predefined hybridization conditions we considerably reduce cross-hybridization problems inherent to the conventional hybridization approach used by Roche and Genomica (such as requiring a 52oC (+/-2) special hybridization buffer). Other important benefits are described below: Market need: The American Cancer Society (ACS) and the American Society of Colposcopy and Cervical Pathology (ASCCP) have issued new guidelines which incorporate DNA testing for high-risk HPV types, along with Pap tests, as a 42 primary screen for women aged 30 and over. The capacity of our probes to detect and also to identify HPV types is clearly an asset if we consider that prevention by vaccine will address only specific HPV types. Multiplex detection: Finding universal experimental conditions for hybridization becomes problematic in multiplex applications where many DNA targets are considered simultaneously. Our method overcomes this problem. Instead of adjusting the hybridization conditions to the probes, a set of probes is selected in such a way as to function under the intended hybridization conditions. Detection of different types in the same patient: According to GeneticLab, the frequency of HPV patient superinfection is 28%, where 71% are infected with 2 subtypes, 21% with 3 subtypes, and 8% with 4 subtypes. Kits that can not discriminate HPV types are unable to differentiate new and persistent infections and have poor diagnostic potential in regard to superinfection. Low production cost and ease of use: A kit based on our technology would require neither expensive instrumentation nor skilled personnel to use. Production of our diagnostic is not expected to be expensive, which would allow the licensor to get a high profit margin. This is crucial for commercial success in the diagnostic field. Even though QIAGEN-Digene presently dominates the HPV DNA diagnostic market and there will be strong competition between new kits—such as the ones from Roche and Genomica—we strongly believe there is an opportunity for these innovative kits to enter this large, underpenetrated, growing market. By fulfilling all the main requirements of the clinical and market needs, our superior kit could quickly become the test of choice for HPV detection. Company Test Name Technology Total number Detection of Comments Market 43 of subtypes detected QIAGEN (Digene) Hybrid Capture 2 RNA-DNA hybridization 2 multiple subtypes in same patient No Ventana Medical Systems Ventana Inform PCR+ In situ hybridization 2 No Genomica CLART® HPV low density 2 microarray 35 Yes Cross-reactivity of probes US, Canada, Europe, Japan, Asia, Australia Europe Greiner Bio One PapilloCheck® DNA-Chip (microarray) 18 high-risk and 6 low-risk types of HPV Yes Cross-reactivity of probes Europe GenoID Reveal HPV 19 not in one assay Semi-quantitative analysis; Very complex Europe and requires RealTime PCR Roche Amplicor PCR+ Molecular beacons PCR + hybridization 13 high-risk types No No individual identification of HPV type; Detects only high risk cases. Roche (LA) Linear Array PCR + hybridization 37 Yes BioMerieux Protect HPVProofer assay (based on NucliSens Easy Q platform of bioMerieux) Detection of oncogenic HPV gene expression 5 high risk types n/d Involves amplification of a portion of the Europe L1 gene by PCR, coding for the major capsid protein, and a subsequent hybridization of the amplification products with the HPV type-specific probes; Unable to distinguish hr-HPV 52 from other high-risk genotypes (33, 35, and 58) presenting 2.2% of all cervical cancers; Less sensitive if a sample has a single infection with some specific HPV genotypes that are poorly amplified by PGMY (HPV 33 and 52); Hybridization probe for HPV 51 not sensitive enough; Requires very controlled temperature of hybridization 52+/-2°C; Presently the most accurate typing kit on the market. Aims at the early detection of cervical Europe carcinogenesis. Technology licensed from the company NorChip in January 2007. The HC2 test uses specific antibodies and chemiluminescent signal amplification to measure the presence of RNA (DNA hybrids formed between a specific RNA probe and the viral DNA). Cross-reactivity of probes; No individual identification of HPV type; Detects only high risk HPV Cytogenetic HPV testing; Allows choice of sample types; No individual identification of HPV type; Poor accuracy US, Canada, Brazil, Europe, Asia, Australia Europe 44 Innogenetics Inno-LiPA HPV Genotyping CE PCR + hybridization QIAGEN (through acquisition of Shenzhen P.G. Biotech Co.) Third Wave Technologies HPV detection PCR -based kit 25 Yes, but have problems with sensitivity, due to the bias induced by 'universal' primers only 4 most n/d common types (6, 11, 16 and 18) No Involves amplification of a portion of the L1 gene by PCR, coding for the major capsid protein, and a subsequent hybridization of the amplification products with the HPV type-specific probes; Problems detecting multiple infections reported, related to typespecific sensitivity of amplification; Full set of HPV types not included; Crosshybridization reported; Requires controlled temperature of hybridization. n/d HPV n/d screening test (14 high-risk types of HPV) Third Wave HPV n/d Technologies genotyping test (HPV—16 and 18) 14 2 Yes n/d GeneticLab PapiPlexTM Multiplex PCR 16 Yes SensiGen AttoSense ™ HPV Test MassArray assay (mass spectrometry coupled with competitive PCR) 15 high risk types n/d Multiplex detection; Complex handling and assay performance, (requires gel manipulation); Only 16 types; not clear how non-including types will perform Ultrasensitive detection kit (1 to 3 copies of HPV DNA in blood or tissue sample). The test is still in development. Licensed from University of Michigan in February 2007 Europe China n/d FDA application for approval expected in 2008-2009 Japan Not on the market Table 7. HPV molecular diagnostic kits 11. COMMERCIALIZATION STRATEGY Our short-term goal is to grant licenses of the HPV test to companies or clinical laboratories. We are in a position to conclude many license agreements within specific fields of use and/or specific territories, since the inventors have developed both, a method for generating specific probes for multiplex diagnostic assay and a specific application of this method which is the HPV assay (both protected by the current patent application). So far we have got serious interest from Warnex, a company based in Québec and Continental Diagnostic, a company based in South Africa. They both would like to get exclusive rights for the HPV assay, in their respective territories. We have also got interest from Dr Gorska at CHUM to implement the HPV assay in their clinical laboratory. It is clear that the clinical validation of the commercial assay will be decisive to consolidate the interest of these groups and to conclude licensing agreement. 45 11.1. Commercialization of the assay in Canada – Warnex partnership Warnex is a life sciences company devoted to protecting public health by providing laboratory services to the pharmaceutical and healthcare sectors. With its three divisions (analytical, bioanalytical and medical laboratory), Warnex provides, to clients in the pharmaceutical and biotechnology industries, a variety of quality control services as well as method development and validation. They also conduct bioavailability and bioequivalence studies for clinical trials, and perform contract R&D. Finally, Warnex provides specialized genetic and biochemical testing for the healthcare industry. This division also has extensive expertise in genetic testing for human identification, molecular diagnostics, and pharmacogenetics. They focus on the development of innovative assays as well as the refinement of existing diagnostic tests to produce assays with greater clinical value and relevance for reliable and cost-effective patient assessment and management. Warnex Medical Laboratories is an important source of specialized testing for hospitals, private laboratories, and medical specialists. As a development-driven medical laboratory, they work extensively in collaboration with medical discovery companies to adapt their research into viable clinical tools for improved diagnosis and monitoring of disease states. Warnex is clearly a relevant partner to bring the HPV assay on the Canadian market. Also, as mentioned in their letter of support, Warnex has accepted to provide in kind contribution for the design of the final format of the assay. Their expertise will also be highly valuable for the regulatory aspect of assay development. 11.2. Commercialization of the assay in Africa – Continental Diagnostic partnership Continental Diagnostics is a Sales and Marketing outfit that would be taking the HPV to the market once the clinical validity of the HPV assay will be established. Their business plan is to boost their current income with the HPV test, which represents a large market in South Africa: cervical cancer accounts for about 25 percent of cancer deaths among black women, there is no formal PAP smear program and women seeking PAP smears in the public health sector are only eligible for one every 10 years after the age of 30. Also the new vaccines are out of reach for most of these women and 15 out of every 100 000 die of Cervical Cancer every year. There are four partners in Continental Diagnostics as presented in Table 8. The company outsources research (validation studies and experience programs) to scientists at the University of Natal who work under Professor Indres Moodley. Prof. Moodley has satellite sites in most African countries through his work with the SA government Department of Science and Technology, where he can conduct validity assays for the HPV test. 46 The company’s customers include pharmacies, private doctors (including specialists and physicians), government and private hospitals, private and government laboratories. Also, the company has a distinct advantage over their competitors in this area as they are a 100% black-owned company and therefore qualify for 60% in the Black Economic Empowerment (BEE) point system that the government uses to award tenders to those who are wholly or partly black-owned. They can also infer points to groups who source their products from us and therefore have a good standing with private companies (laboratories and hospitals) that source from us. Co-funding for the MDEIE project will come from the Industrial Development Corporation in SA, whose brief from the government is to partner African entrepreneurs who create sustainable businesses and jobs in South Africa. Continental Diagnostics Members Masikana Millan Mdleleni.PhD,MBA,LLB Mbulelo Godffery Tabata. Med.Tech Samuel Ndaxola Nkalashe.Med.Tech,MAP Indres Moodley.PhD Expertise Biochemistry Research and Development Responsibility Business Evaluation and Legal Counsel Sales Management Sales Director New Business Development and Marketing Product Research Product Management Pharmaceutical Research Table 8. Continental Diagnostics 11.3. Other partnership and future development Business development activities carried by Anne-Marie Larose at Univalor already bring business relations with two relevant potential industrial partners, as described in the previous section. Ms Larose will continue seeking for companies that would be interested to commercialize the HPV kit in their own territory and/or to develop a new diagnostic kit using this innovative method of generating highly specific probes for multiplex assay. Worthwhile to mention, the proposed project focuses on the clinical validation of an HPV assay for the most frequent HPV types. This will be the first generation of our HPV assay. If we get success in developing a specific and sensitive assay and conclude agreement with at least one company for the commercialization of the assay, we will certainly pursue the development of a second generation of the HPV assay, to diagnose all types of HPV, since we already have probes for 39 types. 12. PRO FORMA BUDGET (in French) 12.1. Coûts du projet de maturation technologique 47 Le budget global requis pour accomplir le projet de recherche et les activités de protection de la propriété intellectuelle est de 200 000$. Les tableaux suivants présentent les budgets requis. R&D (salaries and material ) and indirect expenses Institution research associate HSJ research assistant CHUM laboratory expenses HSJ laboratory expenses CHUM External (Outsource) Phase 1 Phase 2 Total 32 weeks 20 weeks 52 semaines 45 000 $ 30 000 $ 75 000 $ 4 500 $ 8 000 $ 12 500 $ 16 000 $ 21 000 $ 37 000 $ 6 000 $ 6 500 $ 12 500 $ 15 000 $ 0$ 15 000 $ Sous-total (RDI) 86 500 $ 65 500 $ 152 000 $ Indirect expenses (on the 30% from external contribution) 7 500 $ 4 500 $ 12 000 $ Total 94 000 $ 70 000 $ 164 000 $ consultant for product design Table 9 - Estimé budgétaire pour la réalisation du projet de R-D - 12 mois La réalisation du projet nécessitera le travail de 2 personnes sous la supervision du Dr Damian Labuda du CHU Ste-Justine et la collaboration du Dr Isabelle Gorska du CHUM (voir section 7). Il est à noter que ces salaires incluent les bénéfices marginaux. Territory Phase 1 Phase 2 32 semaines 20 semaines Total End of the project 52 semaines Canada 3 000 $ 3 000 $ US 6 500 $ 6 500 $ South Africa 5 500 $ 5 500 $ ARIPO 6 000 $ 6 000 $ 15 000 $ 15 000 $ (16 000 $) (16 000 $) Other territory (Europe, Brazil and/or Angola) Contribution of Univalor for patent cost Reimbursement of Univalor for patent cost Total 20 000 $ 0$ 16 000 $ 16 000 $ 16 000 $ 36 000 $ Table 10 - Estimé budgétaire pour la protection de la propriété intellectuelle 48 Un budget de 36 000$ est requis pour poursuivre le maintient de la propriété intellectuelle dans les différents territoires. Pour ne pas réduire le budget de recherche dans la première phase du projet de recherche, Univalor va supporter des dépenses de brevet de 16 000$, qui lui seront toutefois rembourser à la fin du projet lorsque nous recevrons la dernière contribution du MDEIE dans le cas ou le projet franchise la phase 2 du projet. 12.2. Montage financier Le montant total requis dans ce projet de maturation technologique est de 200 000 $. Une partie de cette somme, c'est-à-dire 164 000$ servira à accorder un contrat de recherche au Dr Damian Labuda du CHU Ste-Justine. Un sous-contrat pour effectuer des travaux dans le laboratoire du Dr Gorska au CHUM est prévu. Le restant du budget servira à défrayer les frais du consultant ainsi que les coûts de protection de la propriété intellectuelle. Source de financement Valorisation-HSJ, société en commandite PMT du MDEIE Total Montant ($) 40 000 160 000 200 000 Table 11 - Montage financier pour financer le projet de maturation technologique 12.3. Documents démontrant la nature des engagements des partenaires financiers La contribution externe au financement du MDEIE, pour un montant de quarante mille dollars (40 000 $), proviendra de Valorisation-HSJ, société en commandite. Cette contribution est conditionnelle aux éléments suivants : 1) l’approbation du financement du projet de recherche annexé à la présente lettre par le MDEIE; 2) la présentation du projet à la satisfaction des membres du Conseil d’administration d’Univalor inc.; 3) la signature d’une option pour négocier des droits exclusifs d’exploitation de la technologie utilisée pour la réalisation du Projet avec notre partenaire Continental Diagnostics. 4) la réception par Valorisation-HSJ de la contribution financière de quarante mille dollars (40 000 $) de notre partenaire Continental Diagnostics; La contribution de Valorisation-HSJ sera décaissée en versements, selon l’avancement du projet, au prorata des ratios de financement requis par le MDEIE et conformément à une entente à intervenir entre le CHU Ste-Justine et Gestion Univalor, sec, au moment de 49 l’acceptation du Projet par le MDEIE. La propriété intellectuelle développée dans le cadre du Projet sera propriété du CHU Ste-Justine, le tout tel que décrit dans les politiques de propriété intellectuelle de l’établissement. Elle sera ensuite cédée à Valorisation-HSJ. Une copie de la lettre signée par Valorisation HSJ, commandité du CHU Ste-Justine, société en commandite est présenté à l’Annexe 4. 12.4. Démonstration que les autres sources de financement possibles ont été prises en considération Le projet soumis dans cette demande ne fait l’objet d’aucune autre source de financement ni d’aucune autre demande de financement. Le financement du projet par le programme de maturation technologique est essentiel pour la réalisation du projet de recherche. 50 13. OUTSIDE OPINIONS 51 REFERENCES Aho, J., Hankins, C., Tremblay, C., Forest, P., Pourreaux, K., Rouah, F. & Coutlee, F. (2004). Genomic polymorphism of human papillomavirus type 52 predisposes toward persistent infection in sexually active women. 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